510 meta confocal microscope

Manufactured by Zeiss

Sourced in Germany, United States, Australia

The Zeiss 510 Meta confocal microscope is a versatile imaging tool designed for advanced microscopy applications. It features a high-resolution optical system, multiple laser excitation options, and sophisticated image acquisition and analysis capabilities. The 510 Meta is capable of producing detailed, high-quality images of a wide range of samples, making it a valuable instrument for applications in various fields of research and study.

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100 protocols using 510 meta confocal microscope

Fluorescent Labeling of Cultured Cells

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Example 4

In a subset of studies, RAECs were labeled with DiI or DiO on the day of recellularization. Briefly, media was removed from a confluent plate of RAECs and replaced with DPBS containing 5 μM SP-DiIC₁₈or SP-DiOC₁₈(Invitrogen). After 5 minutes of incubation at 37° C., plates were transferred to a refrigerator and incubated for 15 minutes at 4° C. Plates were then washed once with PBS and allowed to recover for 2 hours at 37° C. in culture media before isolation and construct seeding. At the end of the experiment, constructs were removed from the bioreactor and imaged on a Stereo Discovery V20 Macro Stereo (Carl Zeiss Inc.), dissected, placed in Slowfade (Invitrogen) and imaged on a 510 Meta Confocal microscope (Carl Zeiss Inc.).

In separate studies, RAECs seeded constructs were labeled with Cell Tracker Green CMFDA (Invitrogen) on the last day of culture (Day 7), by removing the complete culture media and circulating serum free CMFDA containing DMEM (Cellgro) for 45 minutes at 37° C. CMFDA containing media was then replaced with complete MCDB-131 and the constructs were incubated for 45 minutes. CMFDA-labeled constructs were then removed from the bioreactor, dissected, placed in Slowfade (Invitrogen) and imaged on a 510 Meta Confocal microscope (Carl Zeiss Inc.).

US11414644B2. Methods of recellularizing a tissue or organ for improved transplantability (2022-08-16). Regents of the University of Minnesota [US]. Inventors: Doris Taylor [US], Stefan M. Kren [US].

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Fluorescent Labeling of Cultured Cells

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Example 4

US10233420B2. Methods of recellularizing a tissue or organ for improved transplantability (2019-03-19). None [US], None [US]. Inventors: Doris Taylor [US], Stefan M. Kren [US].

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Immunohistochemical Analysis of Embryonic Proteins

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Embryos were collected by gentle centrifugation and fixed in 2% paraformaldehyde in PBS for 15 min, washed 3 times in PBST, and incubated in 4% sheep serum and 1 mg/ml BSA in PBST for 30 min. Embryos were then incubated with a primary antibody (anti-SpMHC, rabbit polyclonal antibody, diluted 1:600, PRIMM, Italy or a commercially available anti-P-Elk1 [Serine 383], mouse monoclonal antibody, dilution 1:100, Santa Cruz Biotechnology, Santa Cruz, USA) overnight at 4°C, washed 4 times in PBST, and followed by another incubation in 4% sheep serum and 1 mg/ml BSA in PBST for 30 min. Similarly, embryos were then incubated with a secondary antibody (anti rabbit-AlexaFluor 555, Invitrogen or anti mouse-HRP) diluted 1:1000 for 1 hr in RT, washed 4 times in PBST, and imaged with a Zeiss 510Meta confocal microscope.

Andrikou C., Pai C.Y., Su Y.H, & Arnone M.I. (2015). Logics and properties of a genetic regulatory program that drives embryonic muscle development in an echinoderm. eLife, 4, e07343.

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Immunofluorescence Analysis of Spheroid Cultures

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Spheroids were harvested by peeling the nylon mesh from the microwells, and collecting the spheroids into Eppendorf tubes. Spheroids were fixed using 4% PFA for 30 minutes at room temperature, followed by permeabilisation using 0.5% Triton X-100 in DPBS for 30 minutes at room temperature. To prevent non-specific binding, 5% bovine serum albumin (Sigma, A7906) was used in the blocking step for 1 hour at room temperature. Cell aggregates were then incubated with primary antibody for Ki67 (Abcam, ab92742) at 1 µg/ml overnight at 4 °C. The anti-rabbit secondary antibody conjugated with Alexa Fluor 594 (Invitrogen; dilution 1:500) was added to the aggregates for 1 hour at room temperature, followed by the nuclear stain, 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), for 30 minutes at room temperature. Stained spheroids were imaged using a Zeiss 510 Meta confocal microscope.

Mosaad E.O., Chambers K.F., Futrega K., Clements J.A, & Doran M.R. (2018). The Microwell-mesh: A high-throughput 3D prostate cancer spheroid and drug-testing platform. Scientific Reports, 8, 253.

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Immunohistochemical Analysis of Mouse Tissues

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Example 16

Mice were anaesthetized, and spleens and livers were collected, frozen in O.C.T. compound and stored at −80° C. until processed. Six to eight micron sections were cut, air dried, fixed for 10 minutes in acetone at −20° C., air dried for at least 4 hours, re-hydrated in TBS for one hour, blocked in 2% N.S. for 1 hour and incubated with primary antibodies overnight at 4° C. with or without 0.1% saponin depending on the antigen. Then, sections were incubated with HRP-labeled secondary antibodies for 1 hour. Slides were developed with ImmPact DAB or NovaRed substrates (Vector Laboratories), air dried, mounted, and analyzed on a Leica microscope. For immunofluorescence stainings, slides were immediately mounted after washing the secondary antibodies. Confocal imaging was done on a Zeiss 510 Meta Confocal microscope.

US10376549B2. Detargeted adenovirus variants and related methods (2019-08-13). None [US], None [US], ADCURE BIOTECHNOLOGIES, LLC. [US]. Inventors: Dmitry M. Shayakhmetov [US], Nelson C. Di Paolo [US].

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Quantifying Mitochondrial Activity in Neurons

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Neurons in 8-chambered slides were incubated with 100 nM Mitotracker Red CMX ROS (Life Technologies) and 3.3 uM of Hoechst 33342 dye (Life Technologies) at 37°C for 20–30 minutes. Cells were rinsed one time in PBS and fixed at 37 C for 15 minutes in 3.7% PFA in neurobasal media. Slides were mounted with ProLong Gold antifade reagent (Life Technologies). The slides were analyzed using a Zeiss 510 Meta confocal microscope. For quantification of mitotracker staining, images were analyzed using Zen blue 2011 software. The display of high magnification images were scaled (which did not change the fluorescence values) so that neurite projections could be observed in all images, including mock-stimulated controls. The profile line tool was then used to draw lines down the center of each neurite, with fluorescent intensity measured each 100 nm to control for different lengths of neurites. Individual lines were drawn for branched neurites. Once all lines were drawn, data tables were generated to show the fluorescent units per 100 nm. Data from multiple images were analyzed for each group and then graphed using Graph-pad Prism to determine differences between groups.

Mukherjee P., Winkler C.W., Taylor K.G., Woods T.A., Nair V., Khan B.A, & Peterson K.E. (2015). SARM1, not MyD88, mediates TLR7/TLR9-induced apoptosis in neurons. Journal of immunology (Baltimore, Md. : 1950), 195(10), 4913-4921.

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Immunocytochemistry Staining of Synapse Markers

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Coverslips were fixed with 4% PFA or ice-cold methanol (95% methanol, 5% HAC) after 49 (direct contact) or 56 (indirect contact) days of differentiation. The immunocytochemistry staining process in short, coverslips were blocked for 1.5 hour at RT in blocking buffer (5% NGS, 0.1% BSA, 0.3% TritonX-100 in PBS 1X) then incubated with primary antibody in blocking buffer for 1 hour at RT followed by overnight incubation at 4°C (primary antibodies are listed in Table A in S1 File). After washes (3X for 5 minutes each in PBS 1X) the coverslips were incubated with secondary antibody in blocking buffer at RT for 1 hour, and mounted with Fluoromount G (Southern Biotech) on glass slides (VWR; Super frost plus). Images were acquired using a Carl Zeiss 510 Meta confocal microscope with 40x (1.2 Numerical Aperture (NA)) and 63x (1.4 NA) oil objectives. Synaptophysin1, VGAT and VGLUT1 puncta analyses were performed with 63x oil objective images using SynD [26 (link)]. A minimum of 10 fields of views (FOVs) was obtained per coverslip. And, synapses were analysed using SynD with threshold parameters of 0.5, 0.7, 1.0 and minimum size of puncta’s 0.7, 0.8, 0.8 μm for Synaptohpysin1, VGAT and VGLUT1, respectively. Immunocytochemistry was performed at RT or primary antibody incubations at 4°C and analysis was performed using Carl Zeiss 510Meta confocal or Leica DM500 B fluorescent microscope.

Nadadhur A.G., Emperador Melero J., Meijer M., Schut D., Jacobs G., Li K.W., Hjorth J.J., Meredith R.M., Toonen R.F., Van Kesteren R.E., Smit A.B., Verhage M, & Heine V.M. (2017). Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells. PLoS ONE, 12(6), e0178533.

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Analyzing Wing Disc Development

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Wing discs were dissected from wandering third instar larve in Grace's medium (source) and incubated for 1 h to incorporate a BrDU analog, EdU, using the Click-iT Kit (Invitrogen). Discs were fixed in 3.5% formaldehyde in PBS, pH 7.2, permeabilized with 0.1% Triton, blocked with 3% BSA, and labeled. The presence of cleaved caspase was detected using Dcp-1 Antibody (Cell Signaling, #9578) at 1/500 dilution, and detected with anti-rabbit Alexa-647 at 1/750. Anti-GFP-FITC (Rockland, #600-402-215) was used at 1/200 dilution and Hoechst 33344 (Life Technologies) at 1/10,000. Discs were mounted in 4% n-propyl gallate in glycerol and imaged on a Zeiss 510 Meta Confocal Microscope. Images were processed by Adobe Photoshop. Total wing disc and the engrailed domain (GFP positive) were defined with the ‘magnetic lasso’ tool. For the measurements shown in Fig. S1E, to reduce background noise, only the top-most epithelial layer corresponding to the wing pouch was measured. For statistics, measurements within genotypes were checked for normality using the shapiro.test() in R. Differences between genotypes were calculated using t.test() in R. All tests used default options.

Daniel S.G., Russ A.D., Guthridge K.M., Raina A.I., Estes P.S., Parsons L.M., Richardson H.E., Schroeder J.A, & Zarnescu D.C. (2017). miR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila. Biology Open, 7(1), bio027391.

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Confocal Microscopy Imaging Protocol

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For RPA stains: Images of immunofluorescent staining were captured on the Zeiss 510 Meta confocal microscope, using three lasers to give excitation at 647, 555 and 488 nM wavelengths. Images at each wavelength were collected sequentially at a resolution of approximately 1024 × 1024 pixels, using the Plan-Apochromat 100×/1.4 Oil objective. All other immunofluorescent staining was imaged using the Leica DM6000B microscope using a HBO lamp with 100W mercury short arc UV bulb light source and four filter cubes, A4, L5, N3 and Y5 to produce excitations at wavelengths 360 488, 555 and 647 nm respectively. Images were captured at each wavelength sequentially using the Plan Apochromat HCX 100×/1.4 Oil objective at a resolution of 1392×1040 pixels.

Densham R.M., Garvin A.J., Stone H.R., Strachan J., Baldock R.A., Daza-Martin M., Fletcher A., Blair-Reid S., Beesley J., Johal B., Pearl L.H., Neely R., Keep N.H., Watts F.Z, & Morris J.R. (2016). Human BRCA1-BARD1 Ubiquitin ligase activity counters chromatin barriers to DNA resection. Nature structural & molecular biology, 23(7), 647-655.

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Dopaminergic Neuron Integrity Assay

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Tyrosine hydroxylase (TH)-Gal4 driven UAS-GFP lines, labeling the neurons with active dopamine synthesis pathway, were crossed with park¹³ flies and progenies were exposed to Sp from 10 days AEL. Twenty days old adult brains from the resultant F1 progeny carrying the genotype TH > Gal4; park¹³, were imaged under Sp treated and untreated conditions to assess the integrity of dopaminergic neurons. Rhabdomere arrangement was visualized in third instar (120 h AEL, Sp fed for 48 h) larval eye imaginal disc by labeling with anti-elav antibody (9F8A9, DSHB). Image analysis was performed through Zeiss 510 META confocal microscope and processed with LSM image browser. Rhabdomere quantification was done through ImageJ. Images were minimally processed for brightness-contrast adjustments using Adobe Photoshop 7.0.

Pradhan P., Majhi O., Biswas A., Joshi V.K, & Sinha D. (2020). Enhanced accumulation of reduced glutathione by Scopoletin improves survivability of dopaminergic neurons in Parkinson’s model. Cell Death & Disease, 11(9), 739.

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